JP6670129B2 - Artificial immunoglobulin fragment composition - Google Patents
Artificial immunoglobulin fragment composition Download PDFInfo
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- JP6670129B2 JP6670129B2 JP2016035400A JP2016035400A JP6670129B2 JP 6670129 B2 JP6670129 B2 JP 6670129B2 JP 2016035400 A JP2016035400 A JP 2016035400A JP 2016035400 A JP2016035400 A JP 2016035400A JP 6670129 B2 JP6670129 B2 JP 6670129B2
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- immunoglobulin
- artificial
- hscfv
- chain variable
- polypeptide
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Description
本発明は、人工ポリクローナル免疫グロブリン組成物、または人工モノクローナル免疫グロブリン組成物に関する。より詳しくは、免疫グロブリンの重鎖可変領域と重鎖定常領域1とヒンジ部分(VH−CH1−hinge)とからなる単鎖可変断片(ScFv、single chain variable fragmentともいう)であって、該重鎖可変領域が互いに異なる複数種の単鎖可変断片を有効成分として含む人工ポリクローナル免疫グロブリン組成物に関する。また、本発明は、本発明に係る人工ポリクローナル免疫グロブリン組成物を投与することを含む、感染症または炎症性疾患の治療方法、並びに本発明に係る人工ポリクローナル免疫グロブリン組成物の、感染症または炎症性疾患の治療用医薬組成物の製造における使用に関する。本発明はまた、任意の数の単鎖可変断片、例えば、ただ1種類の単鎖可変断片を含み得る、人工免疫グロブリン断片組成物に関する。 The present invention relates to an artificial polyclonal immunoglobulin composition or an artificial monoclonal immunoglobulin composition. More specifically, it is a single chain variable fragment (ScFv, single chain variable fragment) comprising a heavy chain variable region, a heavy chain constant region 1 and a hinge portion (VH-CH1-hinge) of an immunoglobulin. The present invention relates to an artificial polyclonal immunoglobulin composition comprising, as an active ingredient, a plurality of single-chain variable fragments having different chain variable regions. Further, the present invention provides a method for treating an infectious disease or an inflammatory disease, which comprises administering the artificial polyclonal immunoglobulin composition according to the present invention, and the infectious disease or inflammation of the artificial polyclonal immunoglobulin composition according to the present invention. Use in the manufacture of a pharmaceutical composition for the treatment of sexually transmitted diseases. The present invention also relates to an artificial immunoglobulin fragment composition, which may comprise any number of single-chain variable fragments, for example, only one single-chain variable fragment.
本願は、2015年2月27日に出願された米国特許出願第14/633,216号への優先権を主張する出願である。当該米国特許出願第14/633,216号は、2011年12月19日に出願された日本特許出願第2011−277163号への優先権を主張して2012年12月17日に出願された米国特許出願第13/716,469号の一部継続出願である。それぞれの特許出願の全体の内容は、本明細書中に参考として援用される。 This application claims priority to US patent application Ser. No. 14 / 633,216 filed Feb. 27, 2015. U.S. Patent Application No. 14 / 633,216 filed on Dec. 17, 2012, claiming priority over Japanese Patent Application No. 2011-277163 filed on Dec. 19, 2011. It is a continuation-in-part of patent application Ser. No. 13 / 716,469. The entire contents of each patent application is incorporated herein by reference.
免疫グロブリン(Ig)は、抗体およびこれと構造上・機能上の関連性のあるタンパク質の総称である。つまり、結合する抗原が明らかになっている免疫グロブリンを、その特定抗原に対応させて抗体と呼んでいる。免疫グロブリンの基本的な分子構造は、軽鎖(L鎖、light chainともいう)と重鎖(H鎖、heavy chainともいう)の大小2種類のポリペプチド2本ずつがジスルフィド結合により連結したものである。重鎖は3つの領域(CH1、CH2、CH3)からなる定常領域(C領域、constant regionともいう)とVH領域からなる可変領域(V領域、variable regionともいう)が連結された構造である。IgMとIgEを除く免疫グロブリンには、CH1とCH2の間にヒンジ(hinge)部分と呼ばれるペプチドが存在する。軽鎖は、CL領域からなる定常領域とVL領域からなる可変領域が連結された構造である。可変領域のアミノ酸配列には多様性があり、これによって多様な抗原に対する多様な抗体が生体内で作られている。 Immunoglobulin (Ig) is a general term for antibodies and proteins that are structurally and functionally related thereto. That is, an immunoglobulin whose binding antigen is known is called an antibody corresponding to the specific antigen. The basic molecular structure of an immunoglobulin is a structure in which two large and small polypeptides, a light chain (also referred to as an L chain and a light chain) and a heavy chain (also referred to as an H chain, a heavy chain), are linked by disulfide bonds. It is. The heavy chain has a structure in which a constant region (C region, also referred to as constant region) composed of three regions (CH1, CH2, CH3) and a variable region (V region, also referred to as variable region) composed of VH region are connected. In immunoglobulins other than IgM and IgE, a peptide called a hinge portion exists between CH1 and CH2. The light chain has a structure in which a constant region composed of a CL region and a variable region composed of a VL region are linked. There is diversity in the amino acid sequences of the variable regions, and as a result, various antibodies against various antigens are produced in vivo.
従来臨床的に使用されてきた免疫グロブリン製剤は、ヒトの血液から抽出した免疫グロブリンを濃縮した血液製剤であり、細菌などの侵入異物と抗原抗体反応を起こし、生体を守る作用を有する。近年、免疫グロブリン製剤は、重症感染症の他に、特発性血小板減少性紫斑病、無ガンマグロブリン血症、川崎病急性期、ギラン・バレー症候群や血管炎である好酸球性多発血管炎性肉芽腫症(Eosinophilic Granulomatosis with Polyangitis:EGPA、旧名 アレルギー性肉芽腫性血管炎、チャーグ・ストラウス症候群)などにも使用されるようになってきている。そして、これらの治療には、静脈を経由して大量に免疫グロブリンを投与する「IVIg治療」がよく用いられている。このIVIg治療は、最近では難治性血管炎などの治療法としても注目されており、その他、国際的にも種々の疾患の治療法として重要視されている。さらに、自己免疫疾患にIVIg治療が認可され、これにともない膠原病や筋無力症を対象として、つぎつぎとIVIg治療が始まっている。また、IVIg治療は川崎病では20年にわたる治療実績があり、最近では2g/kg体重の単回投与がより有効であることで認可されている。以上のように、IVIgは重症度の高い疾患や原因不明の難治性疾患にきわめて有効である。また、IVIgは副作用がほとんどない点でも有用な治療法である。 BACKGROUND ART Immunoglobulin preparations conventionally used clinically are blood preparations obtained by concentrating immunoglobulins extracted from human blood, and have an effect of protecting the living body by causing an antigen-antibody reaction with invading foreign substances such as bacteria. In recent years, immunoglobulin preparations have been used in addition to severe infectious diseases, idiopathic thrombocytopenic purpura, agammaglobulinemia, acute Kawasaki disease, eosinophilic polyangiitis such as Guillain-Barre syndrome and vasculitis. It has also been used for granulomatosis (Eosinophilic Granulomatosis with Polyangitis: EGPA, formerly known as allergic granulomatous vasculitis and Churg-Strauss syndrome). For these treatments, "IVIg treatment" for administering a large amount of immunoglobulin via a vein is often used. This IVIg treatment has recently attracted attention as a treatment method for intractable vasculitis and the like, and is also regarded as important as a treatment method for various diseases internationally. Furthermore, IVIg treatment has been approved for autoimmune diseases, and IVIg treatment has begun one after another for collagen diseases and myasthenia. In addition, IVIg treatment has a track record of treating Kawasaki disease for 20 years, and has recently been approved as a single dose of 2 g / kg body weight being more effective. As described above, IVIg is extremely effective for diseases of high severity and incurable diseases of unknown cause. IVIg is also a useful treatment because it has few side effects.
免疫グロブリン製剤の作用機序については、いくつかの仮説がある。一つの仮説は、免疫グロブリン製剤に含まれている、未知の抗原に対する抗体を含めた多種類の抗体が薬理効果を発揮しているという説である。また別の仮説は、多種類の抗体のうちのミエロペルオキシダーゼ(MPO)に対する抗体(抗MPO抗体)にその効果があり、特にMPOの広範なエピトープに対する多種類の抗MPO抗体が薬理効果を発揮しているという説である。両説において共通していることは、免疫グロブリン製剤が多様な免疫グロブリンの混合物であること、すなわちポリクローナルなものであることが治療効果に大きく寄与しているということである。その他にも仮説があるが、例示した2つの仮説はいずれも有力なものである。 There are several hypotheses about the mechanism of action of immunoglobulin preparations. One hypothesis is that many types of antibodies contained in immunoglobulin preparations, including antibodies against unknown antigens, exert pharmacological effects. Another hypothesis is that among various antibodies, antibodies against myeloperoxidase (MPO) (anti-MPO antibodies) have the effect, and in particular, various anti-MPO antibodies against a wide range of epitopes of MPO exert pharmacological effects. It is the theory that it is. What is common to both theories is that the immunoglobulin preparation is a mixture of various immunoglobulins, that is, polyclonal, which greatly contributes to the therapeutic effect. There are other hypotheses, but both of the two hypotheses illustrated are influential.
現在、臨床的に使用されている免疫グロブリン製剤は血液製剤であるため、原料に由来するウイルスなどの未知の病原体が混入するリスクが常に存在する。事実、病原ウイルスに汚染された血液製剤によって薬害が起こり、大きな社会問題となっている、さらに、対応疾患の増大とともに原料となる血液が不足することも予想されており、血液製剤の免疫グロブリン製剤は安定供給にも不安がある。 At present, since immunoglobulin preparations used clinically are blood preparations, there is always a risk of contamination by unknown pathogens such as viruses derived from raw materials. In fact, blood products contaminated with pathogenic viruses have caused phytotoxicity, which has become a major social problem.Furthermore, it is anticipated that there will be a shortage of blood as a raw material along with an increase in corresponding diseases. Is concerned about stable supply.
このような状況において、患者の感染リスクを減少させ、かつ免疫グロブリン製剤の治癒機転を明らかにする上でも、人工合成品の免疫グロブリン製剤が求められている。人工免疫グロブリン製剤の製造のために、免疫グロブリンの遺伝子を取得し、組換えDNA技術を用いて該遺伝子を発現させ、純化した免疫グロブリンを取得する方法が行われている。例えば、免疫グロブリンの可変領域のみをマウス由来のものに置き換えたキメラ型抗体(特許文献1)や、可変領域の中のCDR領域のみをマウス由来のものに置き換えたヒト化抗体(特許文献2)が、組換えDNA技術によって製造可能である。キメラ型抗体やヒト化抗体は抗体医薬としてすでに実用化されている。また、免疫グロブリン遺伝子を宿主細胞内で可溶状態の正常型タンパク質として発現させる技術として、免疫グロブリンをシャペロニンとの融合タンパク質として発現させる例も知られている(特許文献3)。しかし、これらはすべて単一分子の免疫グロブリン、すなわちモノクローナルの免疫グロブリンである。また、cDNAクローンの性状が不安定であることから、精製品の品質が不安定であり、免疫グロブリン製剤としての治療効果や品質管理上に問題がある。 Under these circumstances, an artificially synthesized immunoglobulin preparation is required to reduce the risk of infection of patients and to clarify the mechanism of healing of the immunoglobulin preparation. In order to produce an artificial immunoglobulin preparation, a method of obtaining a gene for an immunoglobulin, expressing the gene using recombinant DNA technology, and obtaining a purified immunoglobulin has been performed. For example, a chimeric antibody in which only the immunoglobulin variable region is replaced with a mouse-derived one (Patent Document 1), and a humanized antibody in which only the CDR region in the variable region is replaced with a mouse-derived antibody (Patent Document 2) Can be produced by recombinant DNA technology. Chimeric antibodies and humanized antibodies have already been put to practical use as antibody drugs. As a technique for expressing an immunoglobulin gene as a soluble normal protein in a host cell, there is also known an example in which an immunoglobulin is expressed as a fusion protein with a chaperonin (Patent Document 3). However, they are all single molecule immunoglobulins, ie, monoclonal immunoglobulins. In addition, since the properties of the cDNA clone are unstable, the quality of the purified product is unstable, and there is a problem in the therapeutic effect and quality control as an immunoglobulin preparation.
血液製剤に代わる人工免疫グロブリン製剤の提供が求められているが、上述のように現在製造されている人工免疫グロブリンはモノクローナルの免疫グロブリンであり、免疫グロブリン製剤としての治療効果や品質管理上に問題がある。 There is a need to provide artificial immunoglobulin products that replace blood products, but as described above, the currently manufactured artificial immunoglobulins are monoclonal immunoglobulins, and there are problems with the therapeutic effect and quality control of immunoglobulin products. There is.
人工免疫グロブリン製剤の効果には、該製剤が多様な免疫グロブリンの混合物であること、すなわちポリクローナルな免疫グロブリンであることが大きく寄与する。ポリクローナル免疫グロブリン混合物の製造は、多様な免疫グロブリン可変領域に対応する複数種の組換えベクターを調製して組換えベクター混合物を調製し、多様な免疫グロブリン可変領域に対応する複数種のポリペプチドを組換え体内で発現させてそれらのポリペプチド混合物を調製することにより実施できる。しかしながら、このような方法で製造された人工ポリクローナル免疫グロブリン混合物には、精製品の性状が一定しないという問題点があった。 The effect of the artificial immunoglobulin preparation greatly depends on the fact that the preparation is a mixture of various immunoglobulins, that is, a polyclonal immunoglobulin. The production of a polyclonal immunoglobulin mixture involves preparing a plurality of recombinant vectors corresponding to various immunoglobulin variable regions to prepare a recombinant vector mixture, and preparing a plurality of polypeptides corresponding to various immunoglobulin variable regions. It can be carried out by expressing them in a recombinant form to prepare a mixture of these polypeptides. However, the artificial polyclonal immunoglobulin mixture produced by such a method has a problem that the properties of the purified product are not constant.
本発明者は、上記問題点を解決すべく、免疫グロブリンの重鎖可変領域と重鎖定常領域1とヒンジ部分(VH−CH1−hinge)とからなる単鎖可変断片(ScFv)をコードする複数種の遺伝子の発現用ベクターの混合物を調製することを含む人工ポリクローナル免疫グロブリンの製造方法を考案した(特許文献4)。該製造方法は、ScFvをコードするただ1種の遺伝子を含むベクターの調製も含み得る。 In order to solve the above-mentioned problems, the present inventors have developed a plurality of single-chain variable fragments (ScFv) encoding a heavy chain variable region, a heavy chain constant region 1 and a hinge portion (VH-CH1-hinge) of an immunoglobulin. A method for producing an artificial polyclonal immunoglobulin that includes preparing a mixture of vectors for the expression of species genes has been devised (Patent Document 4). The method of manufacture can also include preparing a vector containing only one gene encoding ScFv.
本発明の課題は、治療効果および安全性が高く、かつ安定的な大量供給が可能な人工ポリクローナル免疫グロブリン組成物を提供することである。本発明の課題は、治療効果および安全性が高く、かつ安定的な大量供給が可能な人工免疫グロブリン断片組成物を提供することである。 An object of the present invention is to provide an artificial polyclonal immunoglobulin composition which has a high therapeutic effect and safety and can be stably supplied in large amounts. An object of the present invention is to provide an artificial immunoglobulin fragment composition having a high therapeutic effect and safety and capable of being stably supplied in a large amount.
本発明者は上記課題を解決すべく、ヒト免疫グロブリンの重鎖可変領域と重鎖定常領域1とヒンジ部分(VH−CH1−hinge)とからなるヒト単鎖可変断片(hScFv)であって、該重鎖可変領域が互いに異なる複数種のhScFvの混合物を、免疫グロブリンを発現している組織または細胞に由来するcDNAから組換えDNA技術を用いて製造し、その効果を血管炎モデルマウスを用いて検証した。その結果、配列表の配列番号1から204に記載されたアミノ酸配列で表される204のポリペプチドの混合物が、血管炎モデルマウスにおける血管炎症状を軽減することを見出した(特許文献5、6)。本発明者はまた、任意の数のhScFv、例えば、ただ1種類のhScFvを含み得る組成物を製造した。その結果、本発明者はまた、ただ1種類のhScFvが血管炎、腎炎、糸球体腎炎、肺炎、皮膚炎、またはそれらの任意の組み合わせを、モデルマウスにおいて十分に緩和し得ることを見出した。 In order to solve the above problems, the present inventor has provided a human single chain variable fragment (hScFv) comprising a heavy chain variable region, a heavy chain constant region 1 and a hinge portion (VH-CH1-hinge) of human immunoglobulin, A mixture of a plurality of types of hScFvs having different heavy chain variable regions is produced from cDNA derived from a tissue or cell expressing immunoglobulin using recombinant DNA technology, and the effect is evaluated using a vasculitis model mouse. Verified. As a result, they have found that a mixture of the polypeptides of 204 represented by the amino acid sequences described in SEQ ID NOs: 1 to 204 in the sequence listing reduces vascular inflammation in vasculitis model mice (Patent Documents 5 and 6). ). The inventor has also produced compositions that can include any number of hScFvs, for example, only one hScFv. As a result, the present inventors have also found that only one type of hScFv can sufficiently alleviate vasculitis, nephritis, glomerulonephritis, pneumonia, dermatitis, or any combination thereof in model mice.
すなわち、本発明は以下に関する:
1.ただ1種類の人工免疫グロブリン単鎖可変断片を有効成分として含み、該人工免疫グロブリン単鎖可変断片が配列番号31に記載されたアミノ酸配列を含むポリペプチドである、人工免疫グロブリン断片組成物、
2.ただ1種類の人工免疫グロブリン単鎖可変断片を有効成分として含み、該人工免疫グロブリン単鎖可変断片が配列番号31に記載されたアミノ酸配列からなるポリペプチドである、人工免疫グロブリン断片組成物、
3.ただ1種類の人工免疫グロブリン単鎖可変断片を有効成分として含み、該人工免疫グロブリン単鎖可変断片が配列番号31に記載されたアミノ酸配列を含むポリペプチドである、感染性疾患または炎症性疾患の治療用医薬組成物、
4.ただ1種類の人工免疫グロブリン単鎖可変断片を有効成分として含み、該人工免疫グロブリン単鎖可変断片が配列番号31に記載されたアミノ酸配列からなるポリペプチドである、感染性疾患または炎症性疾患の治療用医薬組成物、
5.前記感染性疾患または炎症性疾患が、血管炎、腎炎、糸球体腎炎、肺炎、皮膚炎、またはそれらの組み合わせである、前項3.または4.の医薬組成物。
That is, the present invention relates to the following:
1. An artificial immunoglobulin fragment composition comprising only one kind of artificial immunoglobulin single chain variable fragment as an active ingredient, wherein the artificial immunoglobulin single chain variable fragment is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31.
2. An artificial immunoglobulin fragment composition, comprising only one kind of artificial immunoglobulin single chain variable fragment as an active ingredient, wherein the artificial immunoglobulin single chain variable fragment is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 31;
3. An infectious disease or an inflammatory disease comprising only one kind of artificial immunoglobulin single chain variable fragment as an active ingredient, wherein the artificial immunoglobulin single chain variable fragment is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31; Therapeutic pharmaceutical composition,
4. An infectious disease or an inflammatory disease comprising only one kind of artificial immunoglobulin single chain variable fragment as an active ingredient, wherein the artificial immunoglobulin single chain variable fragment is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 31; Therapeutic pharmaceutical composition,
5. 2. The above item 3, wherein the infectious disease or inflammatory disease is vasculitis, nephritis, glomerulonephritis, pneumonia, dermatitis, or a combination thereof. Or 4. Pharmaceutical composition.
本発明によれば、免疫グロブリンを発現している組織または細胞に由来するcDNAから組換えDNA技術を用いて製造された、免疫グロブリンの重鎖可変領域と重鎖定常領域1とヒンジ部分(VH−CH1−hinge)とからなるScFv、例えばhScFvであって、該重鎖可変領域が互いに異なる複数種のScFv、例えばhScFvを有効成分として含む人工ポリクローナル免疫グロブリン組成物を提供できる。例えば、配列表の配列番号1から204に記載されたアミノ酸配列で表される204のポリペプチドを有効成分として含む人工ポリクローナル免疫グロブリン組成物を提供できる。 According to the present invention, a heavy chain variable region, a heavy chain constant region 1 and a hinge portion (VH) of an immunoglobulin produced from a cDNA derived from a tissue or a cell expressing the immunoglobulin using recombinant DNA technology. -CH1-hinge) and an artificial polyclonal immunoglobulin composition comprising, as an active ingredient, a plurality of ScFvs having different heavy chain variable regions, for example, hScFv. For example, it is possible to provide an artificial polyclonal immunoglobulin composition containing, as an active ingredient, the polypeptide of 204 represented by the amino acid sequence shown in SEQ ID NOs: 1 to 204 in the sequence listing.
本発明に係る人工ポリクローナル免疫グロブリン組成物は、血液由来の免疫グロブリン製剤と同様に多様の免疫グロブリンを含んでいる。したがって、本発明に係る人工ポリクローナル免疫グロブリン組成物は、治療効果が高く、さらに感染リスクが極めて低いために安全性が高く、かつ安定的な大量供給が可能である。そのため、本発明に係る人工ポリクローナル免疫グロブリン組成物は、IVIg治療に極めて有用である。 The artificial polyclonal immunoglobulin composition according to the present invention contains various immunoglobulins as well as blood-derived immunoglobulin preparations. Therefore, the artificial polyclonal immunoglobulin composition according to the present invention has a high therapeutic effect and has a very low risk of infection, so that it is highly safe and can be stably supplied in large quantities. Therefore, the artificial polyclonal immunoglobulin composition according to the present invention is extremely useful for IVIg treatment.
本発明によれば、免疫グロブリンを発現している組織または細胞に由来するcDNAから組換えDNA技術を用いて製造された人工免疫グロブリン断片組成物を提供できるが、該組成物は有効成分として任意の数のScFv、例えばhScFvを含み、該ScFvはそれぞれ免疫グロブリンの重鎖可変領域と重鎖定常領域1とヒンジ部分(VH−CH1−hinge)とから構成され、該重鎖可変領域は互いに異なるものである。例えば、配列表の配列番号1から204に記載されたアミノ酸配列で表される204のポリペプチドの任意の組み合わせ、任意の部分的組み合わせ、またはただ1つを有効成分として含む人工免疫グロブリン断片組成物を提供することができる。例えば、前記ただ1つのポリペプチドは、配列番号31のアミノ酸を含む、または、該アミノ酸からなるものであり得る。 According to the present invention, an artificial immunoglobulin fragment composition produced by using recombinant DNA technology from cDNA derived from a tissue or cell expressing an immunoglobulin can be provided. Of ScFvs, for example hScFv, each comprising an immunoglobulin heavy chain variable region, a heavy chain constant region 1 and a hinge portion (VH-CH1-hinge), wherein the heavy chain variable regions are different from each other. Things. For example, an artificial immunoglobulin fragment composition containing any combination, any partial combination, or only one of the polypeptides of 204 represented by the amino acid sequences set forth in SEQ ID NOs: 1 to 204 in the sequence listing as an active ingredient Can be provided. For example, the single polypeptide may comprise or consist of the amino acids of SEQ ID NO: 31.
本発明に係る人工免疫グロブリン断片組成物は、血液由来の免疫グロブリン製剤中にみられるような複数の免疫グロブリンを代理するものである。したがって、本発明に係る人工免疫グロブリン断片組成物は、治療効果が高く、さらに感染リスクが極めて低いために安全性が高く、かつ安定的な大量供給が可能である。そのため、本発明に係る人工免疫グロブリン断片組成物は、IVIg治療に極めて有用である。 The artificial immunoglobulin fragment composition according to the present invention substitutes a plurality of immunoglobulins as found in a blood-derived immunoglobulin preparation. Therefore, the artificial immunoglobulin fragment composition according to the present invention has a high therapeutic effect and has a very low risk of infection, so that it can be safely supplied and can be stably supplied in large quantities. Therefore, the artificial immunoglobulin fragment composition according to the present invention is extremely useful for IVIg treatment.
本発明は、人工ポリクローナル免疫グロブリン組成物に関する。より詳しくは、本発明は、免疫グロブリンの重鎖可変領域と重鎖定常領域1とヒンジ部分(VH−CH1−hinge)とからなる単鎖可変断片(ScFv)であって、該重鎖可変領域が互いに異なる複数種の単鎖可変断片を有効成分として含む人工ポリクローナル免疫グロブリン組成物に関する。 The present invention relates to artificial polyclonal immunoglobulin compositions. More specifically, the present invention relates to a single-chain variable fragment (ScFv) comprising an immunoglobulin heavy-chain variable region, heavy-chain constant region 1, and hinge portion (VH-CH1-hinge), The present invention relates to an artificial polyclonal immunoglobulin composition comprising, as an active ingredient, a plurality of types of single-chain variable fragments different from each other.
本発明はまた、人工免疫グロブリン断片組成物に関する。より詳しくは、本発明は、複数種の任意の数の単鎖可変断片(ScFv)であって、それぞれが免疫グロブリンの重鎖可変領域と重鎖定常領域1とヒンジ部分(VH−CH1−hinge)とで構成され、該重鎖可変領域が互いに異なる単鎖可変断片を有効成分として含む人工免疫グロブリン断片組成物に関する。例えば、該人工免疫グロブリン断片組成物は、ただ1種のScFvまたは単一のScFvを含有し得る。本発明に係る人工免疫グロブリン断片組成物は、好ましくはただ1種のhScFvまたは単一のhScFvを含有するものであり得る。 The present invention also relates to an artificial immunoglobulin fragment composition. More specifically, the present invention relates to a plurality of arbitrary numbers of single-chain variable fragments (ScFv), each of which comprises a heavy-chain variable region, a heavy-chain constant region 1 and a hinge portion (VH-CH1-hinge) of an immunoglobulin. ), Wherein the heavy chain variable regions are different from each other. For example, the artificial immunoglobulin fragment composition may contain only one ScFv or a single ScFv. The artificial immunoglobulin fragment composition according to the present invention may preferably contain only one hScFv or a single hScFv.
ポリクローナル免疫グロブリン組成物は、抗原特異性の異なる複数種の免疫グロブリンを含む組成物を意味する。免疫グロブリン断片組成物は、抗原特異性の異なる複数種の任意の数の免疫グロブリンを含む組成物を意味する。例えば、組成物は、特定の抗原特異性を有するただ1種類の免疫グロブリンを含み得る。特定の抗原特異性を有するただ1種類の免疫グロブリンを含有する組成物を、モノクローナル免疫グロブリン組成物と称することがある。 By polyclonal immunoglobulin composition is meant a composition comprising multiple immunoglobulins of different antigen specificity. By immunoglobulin fragment composition is meant a composition comprising any number of multiple immunoglobulins of different antigen specificity. For example, a composition can include only one immunoglobulin having a particular antigen specificity. A composition containing only one immunoglobulin having a particular antigen specificity may be referred to as a monoclonal immunoglobulin composition.
本発明に係る人工ポリクローナル免疫グロブリン組成物は、好ましくは、配列表の配列番号1から204に記載されたアミノ酸配列で表される204のポリペプチドを有効成分として含む人工ポリクローナル免疫グロブリン組成物である。本ポリペプチドは、ヒトガンマグロブリンの重鎖可変領域と重鎖定常領域1とヒンジ部分(VH−CH1−hinge)とからなるヒト単鎖可変断片(hScFv)であって、健常成人末梢血単核球から抽出したRNAから組換えcDNA技術を使用して製造されたポリペプチドである。本発明に係る人工免疫グロブリン断片組成物は、好ましくは、配列番号1から204に記載されたアミノ酸配列で表される204のポリペプチドの少なくとも1つ、例えば配列番号31に記載されたアミノ酸配列で表されるポリペプチドを有効成分として含む人工ポリクローナル免疫グロブリン組成物である。本発明に係る人工免疫グロブリン断片組成物は、また、配列番号31に記載されたアミノ酸配列を含むポリペプチド、好ましくは配列番号31に記載されたアミノ酸配列からなるポリペプチドを有効成分として含む人工ポリクローナル免疫グロブリン組成物であり得る。さらに、本発明に係る人工免疫グロブリン断片組成物は、ただ1種類のScFvを含む人工免疫グロブリン断片組成物であって、ここで、該ScFvが好ましくは配列番号1から204に記載されたアミノ酸配列で表される204のポリペプチドから選ばれる1つのポリペプチド、好ましくは、配列番号31に記載されたアミノ酸配列を含むポリペプチド、より好ましくは配列番号31に記載されたアミノ酸配列からなるポリペプチドである、人工免疫グロブリン組成物であり得る。 The artificial polyclonal immunoglobulin composition according to the present invention is preferably an artificial polyclonal immunoglobulin composition containing, as an active ingredient, the polypeptide of 204 represented by the amino acid sequence set forth in SEQ ID NOs: 1 to 204 in the sequence listing. . The polypeptide is a human single-chain variable fragment (hScFv) comprising a heavy chain variable region of human gamma globulin, a heavy chain constant region 1 and a hinge portion (VH-CH1-hinge), and is a healthy adult peripheral blood mononuclear cell. A polypeptide produced from RNA extracted from a sphere using recombinant cDNA technology. The artificial immunoglobulin fragment composition according to the present invention preferably has at least one of the polypeptides of 204 represented by the amino acid sequence set forth in SEQ ID NOs: 1 to 204, for example, the amino acid sequence set forth in SEQ ID NO: 31. It is an artificial polyclonal immunoglobulin composition containing the expressed polypeptide as an active ingredient. The artificial immunoglobulin fragment composition according to the present invention is also an artificial polyclonal comprising, as an active ingredient, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 31, preferably a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 31 It can be an immunoglobulin composition. Further, the artificial immunoglobulin fragment composition according to the present invention is an artificial immunoglobulin fragment composition containing only one type of ScFv, wherein the ScFv is preferably an amino acid sequence represented by SEQ ID NOS: 1 to 204. A polypeptide selected from the 204 polypeptides represented by the following, preferably a polypeptide comprising the amino acid sequence of SEQ ID NO: 31, more preferably a polypeptide comprising the amino acid sequence of SEQ ID NO: 31 Some may be artificial immunoglobulin compositions.
本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物は、必要に応じて、医薬用に許容される担体(医薬用担体)を含む医薬組成物として製造できる。 The artificial polyclonal immunoglobulin composition and / or artificial immunoglobulin fragment composition according to the present invention can be produced as a pharmaceutical composition containing a pharmaceutically acceptable carrier (pharmaceutical carrier), if necessary.
医薬用担体は、製剤の使用形態に応じて通常使用される、充填剤、増量剤、結合剤、付湿剤、崩壊剤、滑沢剤、希釈剤および賦形剤を例示できる。これらは得られる製剤の投与形態に応じて適宜選択して使用される。より具体的には、水、医薬的に許容される有機溶剤、コラーゲン、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、アルギン酸ナトリウム、水溶性デキストラン、カルボキシメチルスターチナトリウム、ペクチン、キサンタンガム、アラビアゴム、カゼイン、ゼラチン、寒天、グリセリン、プロピレングリコール、ポリエチレングリコール、ワセリン、パラフィン、ステアリルアルコール、ステアリン酸、ヒト血清アルブミン、マンニトール、ソルビトール、ラクトースを例示できる。これらは、本薬剤の剤形に応じて適宜1種類または2種類以上を組み合わせて使用される。そのほか、安定化剤、殺菌剤、緩衝剤、等張化剤、キレート剤、界面活性剤、およびpH調整剤などを適宜使用することもできる。安定化剤は、例えばヒト血清アルブミンや通常のL−アミノ酸、糖類、セルロース誘導体を例示できる。L−アミノ酸は、特に限定はなく、例えばグリシン、システイン、グルタミン酸などのいずれでもよい。糖類も特に限定はなく、例えばグルコース、マンノース、ガラクトース、果糖などの単糖類、マンニトール、イノシトール、キシリトールなどの糖アルコール、ショ糖、マルトース、乳糖などの二糖類、デキストラン、ヒドロキシプロピルスターチ、コンドロイチン硫酸、ヒアルロン酸などの多糖類などおよびそれらの誘導体などのいずれでもよい。セルロース誘導体も特に限定はなく、メチルセルロース、エチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロースナトリウムなどのいずれでもよい。界面活性剤も特に限定はなく、イオン性界面活性剤および非イオン性界面活性剤のいずれも使用できる。界面活性剤には、例えばポリオキシエチレングリコールソルビタンアルキルエステル系、ポリオキシエチレンアルキルエーテル系、ソルビタンモノアシルエステル系、脂肪酸グリセリド系などが包含される。緩衝剤は、ホウ酸、リン酸、酢酸、クエン酸、ε−アミノカプロン酸、グルタミン酸および/またはそれらに対応する塩(例えばそれらのナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩などのアルカリ金属塩やアルカリ土類金属塩)を例示できる。等張化剤は、塩化ナトリウム、塩化カリウム、糖類、グリセリンを例示できる。キレート剤は、例えばエデト酸ナトリウム、クエン酸を例示できる。 Examples of the pharmaceutical carrier include a filler, a bulking agent, a binder, a humectant, a disintegrant, a lubricant, a diluent and an excipient, which are usually used depending on the use form of the preparation. These are appropriately selected and used depending on the administration form of the obtained preparation. More specifically, water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, Examples include gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactose. These may be used alone or in combination of two or more depending on the dosage form of the drug. In addition, a stabilizer, a bactericide, a buffer, a tonicity agent, a chelating agent, a surfactant, a pH adjuster, and the like can be appropriately used. Examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, and cellulose derivatives. The L-amino acid is not particularly limited, and may be, for example, any of glycine, cysteine, glutamic acid and the like. The saccharides are not particularly limited, for example, glucose, mannose, galactose, monosaccharides such as fructose, mannitol, inositol, sugar alcohols such as xylitol, sucrose, maltose, disaccharides such as lactose, dextran, hydroxypropyl starch, chondroitin sulfate, Any of polysaccharides such as hyaluronic acid and derivatives thereof may be used. The cellulose derivative is also not particularly limited, and may be any of methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and the like. The surfactant is not particularly limited, and either an ionic surfactant or a nonionic surfactant can be used. The surfactant includes, for example, polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type, sorbitan monoacyl ester type, fatty acid glyceride type and the like. Buffers include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid, and / or their corresponding salts (eg, alkali metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt and the like). Alkaline earth metal salts). Examples of the tonicity agent include sodium chloride, potassium chloride, sugars, and glycerin. Examples of the chelating agent include sodium edetate and citric acid.
本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物に含まれる有効成分の量は、広範囲から適宜選択される。通常約0.00001−70重量%、好ましくは0.0001−5重量%程度の範囲である。 The amount of the active ingredient contained in the artificial polyclonal immunoglobulin composition and / or the artificial immunoglobulin fragment composition according to the present invention is appropriately selected from a wide range. Usually, it is in the range of about 0.00001-70% by weight, preferably about 0.0001-5% by weight.
本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物の用量範囲は特に限定されず、含有される成分の有効性、投与形態、投与経路、疾患の種類、対象の性質(体重、年齢、病状および他の医薬の使用の有無など)、および担当医師の判断などに応じて適宜選択される。一般的には適当な用量は、例えば対象の体重1kgあたり0.01μg乃至100mg程度、好ましくは約0.1μg乃至1mg程度の範囲である。しかしながら、当該分野においてよく知られた最適化のための一般的な常套的実験を用いてこれらの用量の変更を行うことができる。上記投与量は1日1回乃至数回に分けて投与することができる。また、必要に応じて、従来行われていたIVIg治療で使用されていた用量を参考にして、大量に投与することも可能である。 The dose range of the artificial polyclonal immunoglobulin composition and / or the artificial immunoglobulin fragment composition according to the present invention is not particularly limited, and the efficacy of the contained components, the administration form, the administration route, the type of the disease, the nature of the subject ( Weight, age, medical condition and the use of other medicines, etc.) and the judgment of the attending physician. In general, a suitable dose will be, for example, in the range of about 0.01 μg to 100 mg, preferably about 0.1 μg to 1 mg per kg of the subject's body weight. However, variations in these dosages can be made using routine experimentation for optimization well known in the art. The above dose can be administered once or several times a day. If necessary, a large amount can be administered with reference to the dose used in the conventional IVIg treatment.
投与経路は、全身投与または局所投与のいずれも選択することができる。この場合、疾患、症状などに応じた適当な投与経路を選択する。本発明に係る薬剤は、経口経路および非経口経路のいずれによっても投与できる。非経口経路としては、通常の静脈内投与、動脈内投与のほか、皮下、皮内、筋肉内などへの投与を挙げることができるが、静脈内投与がより好ましい。特に、IVIg治療で大量に投与する場合は、静脈内投与により投与されることが好ましい。 The administration route can be selected from either systemic administration or local administration. In this case, an appropriate administration route is selected according to the disease, symptom and the like. The medicament according to the present invention can be administered by either the oral route or the parenteral route. Examples of the parenteral route include normal intravenous administration and intraarterial administration, as well as subcutaneous, intradermal and intramuscular administration, with intravenous administration being more preferred. In particular, when administering a large amount for IVIg treatment, intravenous administration is preferred.
剤形は、特に限定されず、種々の剤形とすることができる。例えば、溶液製剤として使用できるほかに、これを凍結乾燥化し保存し得る状態にした後、用時、水や生理的食塩水などを含む緩衝液などで溶解して適当な濃度に調製した後に使用することもできる。また持続性剤形または徐放性剤形であってもよい。 The dosage form is not particularly limited, and can be various dosage forms. For example, in addition to being usable as a solution preparation, it can be freeze-dried to make it storable, then used and adjusted to an appropriate concentration by dissolving it in a buffer containing water or physiological saline before use. You can also. It may be a sustained release form or a sustained release form.
具体的には、非経口剤としては、例えば、静脈内注射剤、皮下注射剤、筋肉内注射剤、腹腔内注射剤などの注射剤、経皮投与または貼付剤、軟膏またはローション、口腔内投与のための舌下剤、口腔貼付剤、並びに経鼻投与のためのエアゾール剤、坐剤とすることができるが、これらに限定されない。経口投与のためには、錠剤、カプセル剤、散剤、顆粒剤、丸剤、液剤、乳剤、懸濁液、溶液剤、酒精剤、シロップ剤、エキス剤、エリキシル剤とすることができる。これらの製剤は、製剤工程において通常用いられる公知の方法により製造することができる。 Specifically, parenteral preparations include, for example, injections such as intravenous injections, subcutaneous injections, intramuscular injections, intraperitoneal injections, transdermal or patch, ointments or lotions, buccal administration For oral administration, as well as aerosols and suppositories for nasal administration, but are not limited thereto. For oral administration, tablets, capsules, powders, granules, pills, solutions, emulsions, suspensions, solutions, alcoholic beverages, syrups, extracts, and elixirs can be used. These preparations can be manufactured by a known method usually used in a preparation process.
注射剤を調製する場合は、上記化合物にpH調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤などを添加し、常法により皮下、筋肉内および静脈内用注射剤を製造することができる。この場合のpH調節剤および緩衝剤としてはクエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウムなどを挙げることができる。安定化剤としてはピロ亜硫酸ナトリウム、エチレンジアミン四酢酸(EDTA)、チオグリコール酸、チオ乳酸などを挙げることができる。局所麻酔剤としては塩酸プロカイン、塩酸リドカインなどを挙げることができる。等張化剤としては、塩化ナトリウム、ブドウ糖などを例示できる。 When preparing an injection, a pH adjuster, a buffer, a stabilizer, a tonicity agent, a local anesthetic, etc. are added to the above compound, and a subcutaneous, intramuscular and intravenous injection is prepared by a conventional method. can do. In this case, examples of the pH adjusting agent and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride. Examples of the tonicity agent include sodium chloride and glucose.
経口用固形製剤を調製する場合は、上記有効成分に賦形剤、必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味剤、矯臭剤などを加えた後、常法により錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤などを製造することができる。そのような添加剤としては、当該分野で一般的に使用されるものでよく、例えば、賦形剤としては、乳糖、白糖、塩化ナトリウム、ブドウ糖、デンプン、炭酸カルシウム、カオリン、微結晶セルロース、珪酸などを、結合剤としては、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドンなどを、崩壊剤としては乾燥デンプン、アルギン酸ナトリウム、カンテン末、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、乳糖などを、滑沢剤としては精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコールなどを、矯味剤としては白糖、橙皮、クエン酸、酒石酸などを例示できる。 When an oral solid preparation is prepared, an excipient and, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, a flavoring agent, etc. are added to the above-mentioned active ingredient, and then a tablet is prepared in a conventional manner. , Coated tablets, granules, powders, capsules and the like. Such additives may be those commonly used in the art, for example, excipients such as lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicate As a binder, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylstarch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone, etc. Disintegrators include dried starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, lactose, etc., and lubricants such as purified talc and stearin Salts, borax, polyethylene glycol and the like, as the corrigent sucrose, orange peel, citric acid, can be exemplified tartaric acid.
経口用液体製剤を調製する場合は、上記化合物に矯味剤、緩衝剤、安定化剤、矯臭剤などを加えて常法により内服液剤、シロップ剤、エリキシル剤などを製造することができる。この場合矯味剤としては上記に挙げられたもので良く、緩衝剤としてはクエン酸ナトリウムなどが、安定化剤としてはトラガント、アラビアゴム、ゼラチンなどを挙げることができる。 When preparing an oral liquid preparation, a flavoring agent, a buffering agent, a stabilizer, a deodorant, and the like can be added to the above compound to produce an oral solution, a syrup, an elixir and the like by a conventional method. In this case, those mentioned above may be used as the flavoring agent, and sodium citrate or the like may be used as the buffer, and tragacanth, gum arabic, gelatin or the like may be used as the stabilizer.
本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物は、免疫グロブリン投与治療、例えばIVIgが奏功する疾患の治療に有用である。このような疾患として、感染症、炎症性疾患、特発性血小板減少性紫斑病、無ガンマグロブリン血症、川崎病急性期、ギラン・バレー症候群、並びに血管炎であるチャーグ・ストラウス症候群など、好ましくは感染症および炎症性疾患、より好ましくは血管炎を例示できる。このような疾患としてまた、腎炎および/または糸球体腎炎を例示できる。さらに、肺炎や皮膚炎などの疾患を例示できる。血管炎は、好酸球性多発血管炎性肉芽腫症(Eosinophilic Granulomatosis with Polyangitis:EGPA、旧名 アレルギー性肉芽腫性血管炎、チャーグ・ストラウス症候群)、顕微鏡的多発血管炎(MPA)、急速進行性糸球体腎炎、血管炎によるびまん性肺胞出血および間質性肺炎、結節性多発動脈炎、多発血管炎性肉芽腫症(GPA、旧名:ウェゲナー肉芽腫症)などを挙げることができる。本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物の適用症はこれら疾患に限定されず、免疫グロブリン製剤が奏功する疾患であればいずれの疾患にも適用できる。また、上記疾患から選択される2つ以上の疾患を組み合わせて発症する症例に適用することができる。 The artificial polyclonal immunoglobulin composition and / or artificial immunoglobulin fragment composition according to the present invention is useful for immunoglobulin administration therapy, for example, for the treatment of diseases in which IVIg is effective. Examples of such diseases include infectious diseases, inflammatory diseases, idiopathic thrombocytopenic purpura, agammaglobulinemia, acute Kawasaki disease, Guillain-Barre syndrome, and Churg-Strauss syndrome, which is vasculitis, preferably Infectious diseases and inflammatory diseases, more preferably vasculitis can be exemplified. Such diseases also include nephritis and / or glomerulonephritis. Furthermore, diseases such as pneumonia and dermatitis can be exemplified. Vasculitis is eosinophilic granulomatosis with Polyangitis (EGPA, formerly known as allergic granulomatous vasculitis, Churg-Strauss syndrome), microscopic polyangiitis (MPA), and rapidly progressive Examples include glomerulonephritis, diffuse alveolar hemorrhage due to vasculitis and interstitial pneumonia, polyarteritis nodosa, polyangiitis granulomatosis (GPA, former name: Wegener's granulomatosis). The indications of the artificial polyclonal immunoglobulin composition and / or the artificial immunoglobulin fragment composition according to the present invention are not limited to these diseases, and can be applied to any disease as long as the immunoglobulin preparation is effective. Further, the present invention can be applied to a case in which two or more diseases selected from the above-mentioned diseases are developed in combination.
本発明はまた、本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物を投与することを含む、感染症または炎症性疾患の治療方法を提供する。 The present invention also provides a method for treating an infectious disease or an inflammatory disease, which comprises administering the artificial polyclonal immunoglobulin composition and / or the artificial immunoglobulin fragment composition according to the present invention.
本発明はさらに、配列表の配列番号1から204に記載されたアミノ酸配列で表されるポリペプチドの、感染症または炎症性疾患の治療用医薬組成物の製造における使用を提供する。複数種の任意の数のポリペプチドを、本発明に係る組成物、方法、および使用に従って、用いることができる。例えば、ただ1種類のペプチド、1〜150種類のポリペプチド、1〜100種類のポリペプチド、1〜75種類のポリペプチド、1〜50種類のポリペプチド、1〜25種類のポリペプチド、1〜10種類のポリペプチド、5種類のポリペプチド、4種類のポリペプチド、3種類のポリペプチド、または2種類のポリペプチドを用いることができる。 The present invention further provides use of the polypeptide represented by the amino acid sequence set forth in SEQ ID NOs: 1 to 204 in the manufacture of a pharmaceutical composition for treating an infectious disease or an inflammatory disease. Any number of polypeptides can be used in accordance with the compositions, methods, and uses of the present invention. For example, only one type of peptide, 1 to 150 types of polypeptide, 1 to 100 types of polypeptide, 1 to 75 types of polypeptide, 1 to 50 types of polypeptide, 1 to 25 types of polypeptide, Ten kinds of polypeptides, five kinds of polypeptides, four kinds of polypeptides, three kinds of polypeptides, or two kinds of polypeptides can be used.
本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物の有効成分であるポリペプチドは、例えば組換えDNA技術を使用して製造できる。具体的にはまず、複数種の該ポリペプチドをコードする複数種の遺伝子を混合し、得られた遺伝子混合物を適当なベクターに接触させて組込み、得られた組換えベクターの混合物を、適当な宿主内にトランスフェクションし、得られた形質転換体を培養して該複数種の遺伝子を発現させ、該培養物からそれら遺伝子によりコードされるポリペプチドの混合物を採取し、精製することにより製造できる。 The polypeptide as an active ingredient of the artificial polyclonal immunoglobulin composition and / or the artificial immunoglobulin fragment composition according to the present invention can be produced using, for example, recombinant DNA technology. Specifically, first, a plurality of types of genes encoding a plurality of the polypeptides are mixed, the obtained gene mixture is brought into contact with an appropriate vector and integrated, and the obtained mixture of recombinant vectors is subjected to an appropriate It can be produced by transfecting into a host, culturing the obtained transformant to express the plurality of genes, collecting a mixture of polypeptides encoded by the genes from the culture, and purifying the mixture. .
ベクターは宿主中で複製可能なものであれば特に限定されず、宿主の種類および使用目的により適宜選択される。ベクターは、天然に存在するものを抽出したもののほか、複製に必要な部分以外のDNAの部分が一部欠落しているものでもよい。代表的なものとして、プラスミド、バクテリオファージおよびウイルス由来のベクターを例示できる。プラスミドとして、大腸菌由来のプラスミド、枯草菌由来のプラスミド、酵母由来のプラスミドなどを例示できる。バクテリオファージとして、λファージなどを例示できる。ウイルス由来のベクターとして、例えばレトロウイルス、ワクシニアウイルス、アデノウイルス、パポバウイルス、SV40、鶏痘ウイルス、および仮性狂犬病ウイルスなどの動物ウイルス由来のベクター、あるいはバキュロウイルスなどの昆虫ウイルス由来のベクターを例示できる。その他、トランスポゾン由来、挿入エレメント由来、酵母染色体エレメント由来のベクターなどを例示できる。あるいは、これらを組合せて作成したベクター、例えばプラスミドおよびバクテリオファージの遺伝学的エレメントを組合せて作成したベクター(コスミドやファージミドなど)を例示できる。 The vector is not particularly limited as long as it can be replicated in the host, and is appropriately selected depending on the type of the host and the purpose of use. The vector may be one obtained by extracting a naturally-occurring one, or may be one lacking a part of DNA other than a part necessary for replication. Representative examples include plasmids, bacteriophages, and vectors derived from viruses. Examples of the plasmid include a plasmid derived from Escherichia coli, a plasmid derived from Bacillus subtilis, and a plasmid derived from yeast. Examples of bacteriophage include λ phage. Examples of virus-derived vectors include vectors derived from animal viruses such as retrovirus, vaccinia virus, adenovirus, papovavirus, SV40, fowlpox virus, and pseudorabies virus, and vectors derived from insect viruses such as baculovirus. In addition, vectors derived from transposons, insertion elements, and yeast chromosome elements can be exemplified. Alternatively, vectors prepared by combining these, for example, vectors (cosmids, phagemids, etc.) prepared by combining genetic elements of plasmids and bacteriophages can be exemplified.
ベクターには、目的遺伝子の機能が発揮されるように遺伝子を組込むことが必要であり、少なくとも目的遺伝子配列とプロモーターとをその構成要素とする。これら要素に加えて、所望によりさらに、複製そして制御に関する情報を担持した遺伝子配列、例えば、リボソーム結合配列、ターミネータ、シグナル配列、エンハンサーなどのシスエレメント、スプライシングシグナル、および選択マーカーなどから選択した1つまたは複数の遺伝子配列を自体公知の方法により組合せてベクターに組込むことができる。選択マーカーとして、例えばジヒドロ葉酸還元酵素遺伝子、アンピシリン耐性遺伝子、ネオマイシン耐性遺伝子などを例示できる。 It is necessary to incorporate a gene into a vector so that the function of the gene of interest is exerted, and at least the sequence of the gene of interest and a promoter are components thereof. In addition to these elements, if desired, one selected from gene sequences that carry information about replication and control, such as ribosome binding sequences, terminators, signal sequences, cis elements such as enhancers, splicing signals, and selection markers. Alternatively, a plurality of gene sequences can be combined into a vector by a method known per se. Examples of the selectable marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene and the like.
ベクターに目的遺伝子配列を組込む方法は、自体公知の方法を適用できる。例えば、目的遺伝子配列を適当な制限酵素により処理して特定部位で切断し、次いで同様に処理したベクターと混合し、リガーゼによって再結合する方法が使用される。あるいは、目的遺伝子配列に適当なリンカーをライゲーションし、これを目的に適したベクターのマルチクローニングサイトへ挿入することによっても、所望の組換えベクターが得られる。 As a method for incorporating the target gene sequence into the vector, a method known per se can be applied. For example, a method is used in which the target gene sequence is treated with an appropriate restriction enzyme, cut at a specific site, then mixed with a similarly treated vector, and religated with ligase. Alternatively, a desired recombinant vector can also be obtained by ligating an appropriate linker to the target gene sequence and inserting this into a multicloning site of a vector suitable for the purpose.
宿主として、原核生物および真核生物のいずれも使用できる。原核生物として、例えば大腸菌(エシェリヒアコリ(Escherichia coli))などのエシェリヒア属、枯草菌などのバシラス属、シュードモナスプチダ(Pseudomonas putida)などのシュードモナス属、リゾビウムメリロティ(Rhizobium meliloti)などのリゾビウム属に属する細菌を例示できる。真核生物として、サッカロミセス・セレビシエ(Saccharomyces cerevisiae)、シゾサッカロミセスポンベ(Schizosaccharomyces pombe)などの酵母、Sf9やSf21などの昆虫細胞、あるいはサル腎由来細胞(COS細胞、Vero細胞)、チャイニーズハムスター卵巣細胞(CHO細胞)、マウスL細胞、ラットGH3細胞、ヒトFL細胞や293EBNA細胞、アフリカツメガエル卵母細胞などの動物細胞を例示できる。 As a host, both prokaryotes and eukaryotes can be used. As prokaryotes, for example, genus Escherichia such as Escherichia coli, genus Bacillus such as Bacillus subtilis, genus Pseudomonas such as Pseudomonas putida, genus Rhizobiumium such as Rhizobiumium Bacteria belonging thereto can be exemplified. Examples of eukaryotes include yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, insect cells such as Sf9 and Sf21, or monkey kidney-derived cells (COS cells, Vero cells) and insect cells such as monkey kidney cells. (CHO cells), mouse L cells, rat GH3 cells, human FL cells, 293EBNA cells, and animal cells such as Xenopus oocytes.
ベクターの宿主細胞への導入は、自体公知の手段が応用でき、例えば成書に記載されている標準的な方法(サムブルック(Sambrook)ら編、「モレキュラークローニング,ア ラボラトリーマニュアル 第2版」、1989年、コールドスプリングハーバーラボラトリー)により実施できる。 The vector can be introduced into a host cell by any means known per se. For example, standard methods described in a textbook (edited by Sambrook et al., “Molecular Cloning, Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor Laboratory).
本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物の有効成分であるポリペプチドは、免疫グロブリンの重鎖可変領域(VH)と重鎖定常領域一部(CH1)とを含む融合タンパク質である。抗体重鎖定常領域はその抗体の安定性に寄与するため、本発明に係るポリペプチドは安定性が高く、より血中半減期が長い人工ポリクローナル免疫グロブリン組成物を提供することができる。 The polypeptide as an active ingredient of the artificial polyclonal immunoglobulin composition and / or the artificial immunoglobulin fragment composition according to the present invention comprises an immunoglobulin heavy chain variable region (VH) and a heavy chain constant region part (CH1). Fusion proteins. Since the antibody heavy chain constant region contributes to the stability of the antibody, the polypeptide according to the present invention can provide an artificial polyclonal immunoglobulin composition having high stability and a longer half-life in blood.
本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物の有効成分であるポリペプチドは、したがって、製造において宿主細胞として大腸菌を使用して製造できるため、大量培養が容易である。本発明に係るポリペプチドは、簡便かつ大量に製造することができるため、安定的な大量供給が可能である。 Since the polypeptide which is an active ingredient of the artificial polyclonal immunoglobulin composition and / or the artificial immunoglobulin fragment composition according to the present invention can be produced using Escherichia coli as a host cell in production, large-scale culture is easy. . Since the polypeptide according to the present invention can be easily and mass-produced, stable large-volume supply is possible.
本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物は、血液由来の免疫グロブリン製剤と同様に多様の免疫グロブリンを含んでいる。したがって、本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物は、従来の免疫グロブリン製剤と同様の効果を有する。また、本発明に係る人工ポリクローナル免疫グロブリン組成物および/または人工免疫グロブリン断片組成物は、人工的に製造されているためにウイルスなどの微生物を含まず、感染リスクが極めて少なく、非常に安全性が高い。 The artificial polyclonal immunoglobulin composition and / or artificial immunoglobulin fragment composition according to the present invention contains various immunoglobulins as well as blood-derived immunoglobulin preparations. Therefore, the artificial polyclonal immunoglobulin composition and / or artificial immunoglobulin fragment composition according to the present invention has the same effects as conventional immunoglobulin preparations. In addition, the artificial polyclonal immunoglobulin composition and / or the artificial immunoglobulin fragment composition according to the present invention does not contain microorganisms such as viruses because they are manufactured artificially, has a very low risk of infection, and is extremely safe. Is high.
以下、実施例を示して本発明をより具体的に説明する。本発明は以下に示す実施例によって何ら限定されるものではない。下記実施例において、免疫グロブリン遺伝子を抽出するために使用した血液試料の採取は全て、インフォームドコンセントを行って血液提供者の意思確認をした後に実施したものである。 Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited by the following examples. In the following examples, all blood samples used for extracting immunoglobulin genes were collected after informed consent was obtained and the blood donor's intention was confirmed.
ヒト人工ガンマグロブリンのクローニングと、その大量培養およびタンパク精製を行った。ヒト人工ガンマグロブリンとして、ヒトガンマグロブリンの重鎖可変領域と重鎖定常領域1とヒンジ部分(VH−CH−hinge)とからなるヒト単鎖可変断片(hScFv)を、特許文献4に記載の方法により製造した。 Cloning of human artificial gamma globulin, mass culture and protein purification were performed. As a human artificial gamma globulin, a human single chain variable fragment (hScFv) comprising a heavy chain variable region of human gamma globulin, a heavy chain constant region 1 and a hinge portion (VH-CH-hinge) is described in Patent Document 4. Manufactured by
1.hScFvのクローニング(図1)
インフォームドコンセントを得た健常成人20人の末梢血より末梢血単核球(mononuclear cell;MNC)を分離し、そこからトータルRNAを常法により抽出してプールした。VHの5´端末およびヒンジ領域の3´端末のコンセンサス配列を用いてプライマーを設定した。トータルRNAを鋳型として、コンセンサスプライマーで逆転写ポリメラーゼ連鎖反応(RT−PCT)を行いVH−CH1−hingeをコードするポリクローナルなcDNAを得た。3´端末にヒスチジン6個をコードする配列を付加した3´端末側プライマーを用いてcDNAの3´端末にヒスチジンタグコード領域を付加するとともに、5´末端には16塩基のベクターとの相同配列を付加した。In−Fusionシステムを用いてpBADベクターに相同入れ替えによってcDNAを組み込み、ヒートショック法により大腸菌を形質転換させた。アンピシリン含有選択培地上で生育できる形質転換した菌のコロニーを1000クローン拾い上げ、個々のクローンからプラスミドDNAを抽出してベクター内に組み込まれたcDNAの塩基配列を、蛍光ターミネータを用いたサンガー法により分析し決定した。また個々のクローンの菌体からタンパク抽出を行い硫酸ドデシルナトリウム−ポリアクリル電気泳動(SDS−PAGE)で展開した後、ウエスタンブロットにより抗ヒトFab抗体を用いて、VH−CH1−hingeがタンパクとして合成されているかを確認した。配列解析からVH−CH1−hingeのポリペプチドを発現できる272クローンから、重複しているクローンや、VH−CH1−hingeの構造が壊れているクローン(異構造クローン21、重複クローン46)を除き、最終的にユニーク配列を持つ204クローンを得た。これらのクローンを混合し組換えタンパクを発現させた。
1. Cloning of hScFv (FIG. 1)
Peripheral blood mononuclear cells (MNC) were separated from the peripheral blood of 20 healthy adults who obtained informed consent, and total RNA was extracted therefrom by a conventional method and pooled. Primers were set using the consensus sequences of the 5 'terminal of the VH and the 3' terminal of the hinge region. Reverse transcription polymerase chain reaction (RT-PCT) was performed with consensus primers using the total RNA as a template to obtain a polyclonal cDNA encoding VH-CH1-hinge. A histidine tag coding region was added to the 3 'terminal of the cDNA using a 3' terminal primer to which a sequence encoding six histidines was added to the 3 'terminal, and a homologous sequence with a 16 base vector was added to the 5' end. Was added. CDNA was integrated into the pBAD vector by homologous replacement using the In-Fusion system, and E. coli was transformed by the heat shock method. 1000 colonies of transformed bacteria capable of growing on ampicillin-containing selective medium were picked, and plasmid DNA was extracted from each clone, and the nucleotide sequence of cDNA incorporated into the vector was analyzed by the Sanger method using a fluorescent terminator. I decided. After protein extraction from the cells of each clone and development by sodium dodecyl sulfate-polyacryl electrophoresis (SDS-PAGE), VH-CH1-hinge was synthesized as a protein using an anti-human Fab antibody by Western blotting. Confirmed that it is. From the 272 clones capable of expressing the VH-CH1-hinge polypeptide from sequence analysis, duplicate clones and clones in which the structure of VH-CH1-hinge is broken (heterostructure clone 21, duplicate clone 46) were removed. Finally, 204 clones having a unique sequence were obtained. These clones were mixed to express the recombinant protein.
2.hScFvの大量培養とタンパク精製
クローニングで得られたVH−CH1−hingeタンパクを発現するクローンを500mlのLB培地に混合して37℃で16時間培養し、最終濃度15%のグリセロールを加え、10mlずつチューブに分注し、−80℃で凍結保存し、マスターミックスシードとして使用した。5LのLB培地に溶解したマスターミックスシードを加え、37℃で6時間培養、OD600が0.4〜0.6になったところで、アラビノースを0.002%になるように添加し発現誘導し、さらに37℃にて16時間培養を行い、遠心により菌体を回収した。得られた菌体をトリス−エチレンジアミン四酢酸(Tris−EDTA)バッファー(pH8.0)に懸濁し、デオキシコール酸およびリゾチームを添加し、37℃で撹拌して菌体を可溶化し、さらにDNaseIを添加して大腸菌のDNAを分解し、超音波処理により十分に可溶化した。この懸濁液を高速遠心し不溶性画分(封入体)を沈殿させ、回収した。この不溶性画分を3M 尿素で洗浄し遠心により回収した後、8M 尿素で懸濁し室温で1昼夜放置し目的タンパクを可溶化した。高速遠心により不溶性画分を除去し、上清をニッケルキレートカラム(His−trap)によるクロマトグラフィーにより目的タンパクを分離した。さらに精製のためニッケルキレートカラム(His−trap)クロマトグラフィーを再度行い高度精製した。
2. Mass culture of hScFv and protein purification Clones expressing the VH-CH1-hinge protein obtained by cloning were mixed with 500 ml of LB medium, cultured at 37 ° C. for 16 hours, glycerol having a final concentration of 15% was added, and 10 ml each was added. Dispensed into tubes, stored frozen at -80 ° C, and used as master mix seeds. A master mix seed dissolved in 5 L of LB medium was added, and cultured at 37 ° C. for 6 hours. When the OD600 reached 0.4 to 0.6, arabinose was added to 0.002% to induce expression. The cells were further cultured at 37 ° C. for 16 hours, and the cells were collected by centrifugation. The obtained cells were suspended in a Tris-ethylenediaminetetraacetic acid (Tris-EDTA) buffer (pH 8.0), deoxycholic acid and lysozyme were added, the mixture was stirred at 37 ° C. to solubilize the cells, and DNaseI was further dissolved. Was added to decompose the DNA of Escherichia coli and sufficiently solubilized by sonication. The suspension was centrifuged at high speed to precipitate and collect an insoluble fraction (inclusion body). The insoluble fraction was washed with 3M urea, collected by centrifugation, suspended in 8M urea, and left at room temperature for 24 hours to solubilize the target protein. The insoluble fraction was removed by high-speed centrifugation, and the target protein was separated from the supernatant by chromatography using a nickel chelate column (His-trap). For further purification, nickel chelate column (His-trap) chromatography was performed again to perform highly purification.
3.エンドトキシンの除去(低減化)
得られた精製タンパクにはエンドトキシンが混入しており、これを除去するために、タンパク溶液を6M グアニジン塩酸を強アルカリにしたバッファーに対し透析し、強アルカリ環境の下に3昼夜放置し、エンドトキシンの加水分解を進め、10k ポアの限外濾過によって分解物を除去し、エンドトキシンの低減化を行った。
3. Endotoxin removal (reduction)
Endotoxin was contaminated in the obtained purified protein. To remove the endotoxin, the protein solution was dialyzed against a buffer in which 6M guanidine hydrochloride was made strongly alkaline, and left under a strong alkaline environment for three days and nights. The hydrolysis of was carried out, and the degradation products were removed by ultrafiltration through a 10 k pore to reduce endotoxin.
4.結果
配列表の配列番号1から204に記載のアミノ酸配列で表される204の異なるポリペプチドをコードする204のクローンが得られた。すなわち、204の異なるhScFvが得られた。204の異なるhScFvの混合物を精製した精製標品に含まれるエンドトキシン濃度は、10〜5ng/1mg hScFvであった。
4. Results 204 clones encoding 204 different polypeptides represented by the amino acid sequences of SEQ ID NOs: 1 to 204 in the sequence listing were obtained. That is, 204 different hScFv were obtained. The endotoxin concentration contained in the purified preparation obtained by purifying a mixture of 204 different hScFvs was 10 to 5 ng / 1 mg hScFv.
実施例1で製造した複数種のhScFvからなる混合物の血管炎に対する治療効果を、血管炎自然発症モデルマウスであるSCG/Kjマウスを使用して検討した。使用したhScFv混合物は、配列表の配列番号1から204に記載のアミノ酸配列で表される204の異なるポリペプチドからなる。hScFv混合物は、0.45M アルギニン(Arg)、0.45% グリシン(Gly)、および0.9% 塩化ナトリウムを含む1.5% D−マンニトールに溶解して使用した。 The therapeutic effect on the vasculitis of a mixture comprising a plurality of types of hScFvs prepared in Example 1 was examined using SCG / Kj mice, which are spontaneous vasculitis model mice. The hScFv mixture used consists of 204 different polypeptides represented by the amino acid sequences set forth in SEQ ID NOs: 1 to 204 in the sequence listing. The hScFv mixture was used dissolved in 1.5% D-mannitol containing 0.45M arginine (Arg), 0.45% glycine (Gly), and 0.9% sodium chloride.
まず、10週齢のSCG/KjマウスにhScFvを10〜40mg/Kg/dayの処方で腹腔内投与(ip)により5日間連続投与し、13週齢になったところで、CO2ガスにて安楽殺し、血管炎の指標である血清中のMPO−ANCA(myeloperoxidase−specific anti−neutrophil cytoplasmic antibody)値、脾臓重量、末梢血中の白血球数、リンパ球数、単球数、顆粒球(好中球)数、および血小板数を測定し、その結果により治療効果を判定した(図2)。 First, the hScFv to 10-week-old SCG / Kj mice for 5 consecutive days by intraperitoneal administration (ip) with the formulation of 10 to 40 mg / Kg / day, upon reaching the age 13, comfort in the CO 2 gas Killing, MPO-ANCA (myeloperoxidase-specific anti-neutrophil cytoplasmic antibody) value in serum, which is an indicator of vasculitis, spleen weight, white blood cell count in peripheral blood, lymphocyte count, monocyte count, granulocyte (neutrophil) ) The number and platelet count were measured, and the therapeutic effect was determined based on the results (FIG. 2).
hScFv混合物の投与により、血管炎の指標である血清中MPO−ANCA値の減少が認められた(図3)。また、脾臓重量の低下傾向が認められ(図4)、さらに、末梢血中の白血球数、リンパ球数、単球数、顆粒球(好中球)数、血小板数が低下した(図5−A、5−B、および5−C)。以上の結果から、hScFv混合物の治療効果が認められた。 Administration of the hScFv mixture resulted in a decrease in serum MPO-ANCA value, which is an indicator of vasculitis (FIG. 3). In addition, a tendency of a decrease in spleen weight was observed (FIG. 4), and further, the numbers of white blood cells, lymphocytes, monocytes, granulocytes (neutrophils), and platelets in peripheral blood decreased (FIG. 5). A, 5-B, and 5-C). From the above results, the therapeutic effect of the hScFv mixture was confirmed.
本発明に係る204のhScFvポリペプチドについてさらなる解析を実施し、SCG/Kjマウスの炎症症状への特定のhScFv断片の相対的有効性を測定した。SCG/Kjマウスは血管炎自然発症モデルマウスであり、急速進行性糸球体腎炎モデルとしても知られている。本実施例で説明するように、ポリペプチドの1つ、すなわち配列番号31のアミノ酸配列を有するポリペプチドがSCG/Kjマウスの腎機能に強力な改善効果、例えば増加した半月体形成率の改善、腎糸球体組織傷害の改善、および肺組織傷害の改善を示した。さらに、該ポリペプチドはまた、SCG/Kjマウスの免疫学的機能に改善効果を示した。これら改善効果に加えて、増加した脾臓重量および脾臓組織切片に認められた異常が改善された。これら改善効果に加えてまた、末梢血中の増加した白血球数、リンパ球数、単球数、および顆粒球数が改善された。これら結果は、該ポリペプチドが腎炎、例えば糸球体腎炎などの炎症性疾患の治療に有効であることを示す。 Further analysis was performed on the 204 hScFv polypeptides of the present invention to determine the relative efficacy of particular hScFv fragments on inflammatory conditions in SCG / Kj mice. The SCG / Kj mouse is a spontaneous vasculitis model mouse, and is also known as a rapidly progressive glomerulonephritis model. As described in this example, one of the polypeptides, ie, a polypeptide having the amino acid sequence of SEQ ID NO: 31, has a strong improving effect on renal function of SCG / Kj mice, for example, an increased rate of crescent formation, It showed an improvement in renal glomerular tissue injury and an improvement in lung tissue injury. Furthermore, the polypeptide also showed an improving effect on the immunological function of SCG / Kj mice. In addition to these improving effects, the increased spleen weight and abnormalities observed in spleen tissue sections were improved. In addition to these improving effects, increased white blood cell counts, lymphocyte counts, monocyte counts, and granulocyte counts in peripheral blood were also improved. These results indicate that the polypeptide is effective in treating inflammatory diseases such as nephritis, eg, glomerulonephritis.
1.材料および方法 1. Materials and methods
[組換えhScFvタンパク質の精製] [Purification of recombinant hScFv protein]
実施例1で製造した204クローンのhScFvライブラリから、複数種の組換えhScFvタンパク質を単離した。204クローンをq群、t群、d群、u群の4群に分け、次いで各群内のクローンの1クローンずつからhScFvを精製した。hScFvタンパク質を血管炎自然発症モデルであるSCG/Kjマウスに腹腔内投与して治療を行った。各クローンの精製タンパク質は次のように単離した。クローンの培養を3L行い、次いで菌体を20 mLのリン酸緩衝生理食塩水(PBS)を用いて超音波破砕後、ニッケルカラムクロマトグラフィー、ブチルセファロースカラムクロマトグラフィー、DEAE−セファロースカラムクロマトグラフィー、コバルト−セファロースカラムクロマトグラフィーで処理し、その後、9% 塩化ナトリウム、1.5% D−マンニトールを含むPBSに置換した。上記手法による検討を繰り返し行って4クローンを選択し、有効な群を得た。 A plurality of types of recombinant hScFv proteins were isolated from the hScFv library of 204 clones produced in Example 1. 204 clones were divided into four groups, q group, t group, d group, and u group, and then hScFv was purified from each clone in each group. The hScFv protein was treated by intraperitoneal administration to SCG / Kj mice, which are spontaneous vasculitis models. The purified protein of each clone was isolated as follows. After culturing the clone for 3 L, the cells were ultrasonically crushed with 20 mL of phosphate buffered saline (PBS), and then nickel column chromatography, butyl sepharose column chromatography, DEAE-sepharose column chromatography, cobalt -Sepharose column chromatography, followed by replacement with PBS containing 9% sodium chloride, 1.5% D-mannitol. The examination by the above method was repeated and 4 clones were selected to obtain an effective group.
[治療の評価] [Evaluation of treatment]
hScFvを10週齢の雌性SCG/Kjマウスに1〜40mg/Kg/dayの濃度で5日間に亘って腹腔内投与(ip)し、13週齢になったところで該マウスをCO2ガスにて安楽殺した。血管炎のバイオマーカである血清中のMPO−ANCA(myeloperoxidase−specific anti−neutrophil cytoplasmic antibody)値、抗モエシン抗体、およびサイトカイン/ケモカイン、脾臓重量、血液細胞中の白血球(WBC)数、リンパ球数、単球数、顆粒球(好中球)数を測定した。 hScFv was administered intraperitoneally (ip) to 10-week-old female SCG / Kj mice at a concentration of 1 to 40 mg / Kg / day for 5 days. At 13 weeks of age, the mice were exposed to CO 2 gas. Euthanized. MPO-ANCA (myeloperoxidase-specific anti-neutrophil cytoplasmic antibody) value in serum which is a biomarker of vasculitis, anti-moesin antibody, cytokine / chemokine, spleen weight, leukocyte count in blood cells, lymphocyte count (WBC) , Monocyte count and granulocyte (neutrophil) count were measured.
さらに、腎臓、肺、および脾臓を10% ホルマリンで固定した後、パラフィン包埋して組織切片をスライド上に作成し、ヘマトキシリンエオジン(HE)染色した。該スライドグラス上の糸球体組織中の最も重要な指標である半月体形成を顕微鏡により観察した。また、該スライドグラス上で肺および脾臓の組織学的観察を行った。 Further, after fixing the kidney, lung and spleen with 10% formalin, a tissue section was prepared by embedding in paraffin on a slide, and stained with hematoxylin and eosin (HE). Crescent formation, the most important indicator in glomerular tissue on the slide glass, was observed with a microscope. Histological observation of the lung and spleen was performed on the slide glass.
統計解析はスチューデントのt−検定により実施した。 Statistical analysis was performed by Student's t-test.
2.結果 2. result
選択した4クローンの中で、クローンQRq01がSCG/Kjマウスにおける炎症の諸症状の低減に最も効果があった。クローンQRq01は配列番号31に記載のポリペプチドをコードする。結果を図6−図13に示す。 Of the four clones selected, clone QRq01 was most effective in reducing various symptoms of inflammation in SCG / Kj mice. Clone QRq01 encodes the polypeptide set forth in SEQ ID NO: 31. The results are shown in FIGS.
図6は、クローンQRq01 hScFv治療により糸球体中の半月体形成が減少したことを示す。糸球体中の半月体形成は、糸球体組織内の血管炎の最も主要な指標であり、そしてSCG/Kjマウスにおいて増加していたが、クローンQRq01 hScFvを1mg/Kg/dayの濃度で5日間に亘り投与することにより有意に減少した。 FIG. 6 shows that clonal QRq01 hScFv treatment reduced crescent formation in glomeruli. Crescent formation in the glomeruli was the most major indicator of vasculitis in glomerular tissue and was increased in SCG / Kj mice, but clone QRq01 hScFv was maintained at a concentration of 1 mg / Kg / day for 5 days. , Significantly decreased.
図7は、クローンQRq01 hScFv治療による腎臓内の糸球体中半月体形成の回復について組織学的観察結果を示す。具体的には、 図7は、クローンQRq01 hScFvを1mg/Kg/dayの濃度で5日間に亘り投与することによる糸球体中半月体形成の評価における腎臓組織切片の顕微鏡観察結果を示す。SCG/Kjマウスにおける半月体形成は、ほとんど正常糸球体の半月体形成といっていい程度に回復した。図7において、「白丸」は糸球体を指す。 FIG. 7 shows histological observations on the recovery of crescent crescent formation in the kidney by treatment with clone QRq01 hScFv. Specifically, FIG. 7 shows the results of microscopic observation of kidney tissue sections in the evaluation of crescent formation in glomeruli by administering clone QRq01 hScFv at a concentration of 1 mg / Kg / day for 5 days. The crescent formation in the SCG / Kj mice almost recovered to the extent of normal glomerular crescent formation. In FIG. 7, "white circle" indicates a glomerulus.
図8は、クローンQRq01 hScFv治療による肺臓内の炎症の回復について組織学的観察結果を示す。具体的には、 図8は、クローンQRq01 hScFvを1mg/Kg/dayの濃度で5日間に亘り投与することによる肺組織切片の組織学的観察結果を示す。hScFv治療をしないコントロールSCG/Kjマウスでは、組織像で血管炎、出血像、およびリンパ濾胞が観察された。hScFv治療により、組織像はほぼ正常を示した。 FIG. 8 shows the results of histological observation on the recovery of inflammation in the lungs by treatment with clone QRq01 hScFv. Specifically, FIG. 8 shows histological observation results of a lung tissue section obtained by administering the clone QRq01 hScFv at a concentration of 1 mg / Kg / day for 5 days. In control SCG / Kj mice not treated with hScFv, vasculitis, bleeding, and lymph follicles were observed in histology. The histology was almost normal by hScFv treatment.
図9−Aおよび9−Bは、クローンQRq01 hScFv治療による末梢血中の白血球数、リンパ球数、単球数、顆粒球(好中球)数を示す。SCG/Kjマウスで増加していた末梢血中の白血球(WBC)数およびリンパ球数(図9−A)、単球数および顆粒球(好中球)数(図9−B)は、クローンQRq01 hScFvを1mg/Kg/dayの濃度で5日間に亘り投与することにより減少した。 FIGS. 9-A and 9-B show the white blood cell count, lymphocyte count, monocyte count, and granulocyte (neutrophil) count in peripheral blood after treatment with clone QRq01 hScFv. Increased numbers of leukocytes (WBC) and lymphocytes in peripheral blood (FIG. 9-A), monocytes and granulocytes (neutrophils) (FIG. 9-B) in SCG / Kj mice were increased by clones. It was reduced by administering QRq01 hScFv at a concentration of 1 mg / Kg / day for 5 days.
図10は、クローンQRq01 hScFv治療による脾臓重量の回復を示す。SCG/Kjマウスで増加していた脾臓重量は、クローンQRq01 hScFvを1mg/Kg/dayの濃度で5日間に亘り投与することにより減少した。 FIG. 10 shows recovery of spleen weight by treatment with clone QRq01 hScFv. The increased spleen weight in SCG / Kj mice was reduced by administering clone QRq01 hScFv at a concentration of 1 mg / Kg / day for 5 days.
図11は、クローンQRq01 hScFv治療による脾臓内の炎症の回復について組織学的観察結果を示す。クローンQRq01 hScFvを1mg/Kg/dayの濃度で5日間に亘り投与することにより、溶媒を投与したコントロールSCG/Kjマウスでは不明瞭であった脾臓内の赤脾髄と白脾髄が明瞭に認められた。 FIG. 11 shows the results of histological observation on recovery of inflammation in the spleen by treatment with clone QRq01 hScFv. By administering the clone QRq01 hScFv at a concentration of 1 mg / Kg / day for 5 days, the red pulp and white pulp in the spleen were unclear in the control SCG / Kj mice to which the solvent was administered. Was done.
図12−Aおよび12−Bは、クローンQRq01 hScFv治療による血清中のMPO−ANCAおよび抗モエシン抗体の減少を示す。SCG/Kjマウスで増加していた血清中のMPO−ANCA(図12−A)および抗モエシン抗体(図12−B)は、クローンQRq01 hScFvを1mg/Kg/dayの濃度で5日間に亘り投与することにより減少した。抗モエシン抗体は、MPO−ANCAと同様、ANCA関連血管炎との関連が注目されており、また、顕微鏡的多発血管炎(MPA)などの血管炎で陽性になることや、皮膚動脈炎の結節性多発動脈炎への移行に伴って上昇することが報告されている。したがって、クローンQRq01 hScFv投与による抗モエシン抗体およびMPO−ANCAの減少結果は、クローンQRq01 hScFvの血管炎治療効果を示すものであると考えることができる。 Figures 12-A and 12-B show the reduction of serum MPO-ANCA and anti-moesin antibodies by treatment with clone QRq01 hScFv. MPO-ANCA (FIG. 12-A) and anti-moesin antibody (FIG. 12-B) in serum increased in SCG / Kj mice were administered with the clone QRq01 hScFv at a concentration of 1 mg / Kg / day for 5 days. It was reduced by doing. Similar to MPO-ANCA, anti-moesin antibody has been attracting attention for its association with ANCA-associated vasculitis. In addition, anti-moesin antibody becomes positive in vasculitis such as microscopic polyangiitis (MPA) and nodules of cutaneous arteritis It has been reported to increase with the transition to polyarteritis polytitis. Therefore, it can be considered that the result of the reduction of the anti-moesin antibody and MPO-ANCA by the administration of clone QRq01 hScFv indicates the therapeutic effect of clone QRq01 hScFv on vasculitis.
図13は、クローンQRq01 hScFv治療による血清中のサイトカイン/ケモカインのレベルの減少を示す。増加していた血清中のサイトカイン/ケモカインのレベルは、クローンQRq01 hScFvを1mg/Kg/dayの濃度で5日間に亘りSCG/Kjマウスに投与することにより減少した。図13において、黒色バーおよび白色バーはそれぞれQRq01クローンを投与したマウス群および溶媒を投与したマウス群を指す。 FIG. 13 shows the reduction of serum cytokine / chemokine levels by clonal QRq01 hScFv treatment. Elevated serum cytokine / chemokine levels were reduced by administering clone QRq01 hScFv at a concentration of 1 mg / Kg / day to SCG / Kj mice for 5 days. In FIG. 13, a black bar and a white bar indicate a mouse group to which the QRq01 clone was administered and a mouse group to which the solvent was administered, respectively.
凡例
1 コントロール(溶媒)
2 クローン=QRq01(1mg/Kg/day)
3 クローン=QRq01p(1mg/Kg/day)
4 クローン=QRq01(4mg/Kg/day)
5 クローン=Rq01(4mg/Kg/day)
6 クローン=QRt01(1mg/Kg/day)
7 天然のIgG(4mg/Kg/day)
8 ネガティブコントロール(1mg/Kg/day)
9 健常コントロール(C57BL/6)
Legend 1 Control (solvent)
2 clones = QRq01 (1 mg / Kg / day)
3 clones = QRq01p (1 mg / Kg / day)
4 clones = Qrq01 (4 mg / Kg / day)
5 clones = Rq01 (4 mg / Kg / day)
6 clones = QRt01 (1 mg / Kg / day)
7 Natural IgG (4 mg / Kg / day)
8 Negative control (1 mg / Kg / day)
9 healthy controls (C57BL / 6)
略称:
IL−1a:インターロイキン−1α(interleukin−1 alpha)
IL−1b:インターロイキン−1β(interleukin−1 beta)
IL−2:インターロイキン−2(interleukin−2)
IL−3:インターロイキン−3(interleukin−3)
IL−4:インターロイキン−4(interleukin−4)
IL−5:インターロイキン−5(interleukin−5)
IL−6:インターロイキン−6(interleukin−6)
IL−9:インターロイキン−9(interleukin−9)
IL−10:インターロイキン−10(interleukin−10)
IL12p40:インターロイキン−12 サブユニット p40(interleukin−12 subunit p40)
IL−12p70:インターロイキン−12 サブユニット p70(interleukin−12 subunit p70)
IL−13:インターロイキン−13(interleukin−13)
IL−17:インターロイキン−17(interleukin−17)
Eotaxin:エオタキシン(eotaxin)
G−CSF:顆粒球コロニー刺激因子(granulocyte colony−stimulating factor)
GM−CSF:顆粒球/マクロファージコロニー刺激因子(granulocyte macrophage−colony stimulating factor)
IFN−g:インターフェロンγ(interferon gamma)
KC:ケラチノサイト由来ケモカイン(keratinocyte−derived chemokine)
MCP−1:単球走化性因子タンパク質−1(monocyte chemoattractant protein−1)
MIP−1a:マクロファージ炎症性タンパク質−1α(macrophage inflammatory protein−1 alpha)
MIP−1b:マクロファージ炎症性タンパク質−1β(macrophage inflammatory protein−1 beta)
RANTES:活性化により制御される、正常T細胞で発現および分泌されるタンパク質(regulated on activation,normal T cell expressed and secreted)
TNF−a:腫瘍壊死因子−α(tumor necrosis factor−alpha)
IL−15:インターロイキン−15(interleukin−15)
IL−18:インターロイキン−18(interleukin−18)
FGF−basic:塩基性−線維芽細胞増殖因子(fibroblast growth factor−basic)
LIF:白血病阻止因子(leukemia−inhibitory factor)
M−CSF:マクロファージコロニー刺激因子(macrophage colony−stimulating factor)
MIG:インターフェロンγ誘導性モノカイン(monokine induced by interferon gamma)
MIP−2:マクロファージ炎症性タンパク質−2(macrophage inflammatory protein−2)
PDGF−bb:血小板由来増殖因子−BB(platelet−derived growth factor−BB)
VEGF:血管内皮増殖因子(vascular endothelial growth factor)
IL−6sR:インターロイキン−6可溶性受容体(interleukin−6 soluble receptor)
IL−23:インターロイキン−23(interleukin−23)
abbreviation:
IL-1a: interleukin-1 alpha (interleukin-1 alpha)
IL-1b: interleukin-1 beta (interleukin-1 beta)
IL-2: interleukin-2
IL-3: interleukin-3
IL-4: interleukin-4
IL-5: interleukin-5 (interleukin-5)
IL-6: interleukin-6
IL-9: interleukin-9
IL-10: interleukin-10
IL12p40: interleukin-12 subunit p40
IL-12p70: interleukin-12 subunit p70
IL-13: interleukin-13
IL-17: interleukin-17
Eotaxin: Eotaxin
G-CSF: granulocyte colony-stimulating factor
GM-CSF: granulocyte / macrophage colony stimulating factor
IFN-g: interferon gamma (interferon gamma)
KC: keratinocyte-derived chemokine
MCP-1: monocyte chemoattractant protein-1
MIP-1a: macrophage inflammatory protein-1α (macrophage inflammation protein-1 alpha)
MIP-1b: macrophage inflammatory protein-1 beta
RANTES: Regulated on activation, normal T cell expressed and secreted, controlled by activation
TNF-a: tumor necrosis factor-α (tumor necrosis factor-alpha)
IL-15: interleukin-15
IL-18: interleukin-18
FGF-basic: basic-fibroblast growth factor-basic
LIF: Leukemia-inhibitory factor
M-CSF: macrophage colony-stimulating factor
MIG: interferon gamma-induced monokine (monokine induced by interferon gamma)
MIP-2: macrophage inflammatory protein-2
PDGF-bb: platelet-derived growth factor-BB
VEGF: Vascular endothelial growth factor
IL-6sR: interleukin-6 soluble receptor
IL-23: interleukin-23
本発明は、感染性疾患、炎症性疾患、特発性血小板減少性紫斑病、無ガンマグロブリン血症、川崎病急性期、ギラン・バレー症候群、並びに血管炎である好酸球性多発血管炎性肉芽腫症(Eosinophilic Granulomatosis with Polyangitis:EGPA、旧名 アレルギー性肉芽腫性血管炎、チャーグ・ストラウス症候群)、顕微鏡的多発血管炎(MPA)、急速進行性糸球体腎炎、血管炎によるびまん性肺胞出血および間質性肺炎、結節性多発動脈炎、多発血管炎性肉芽腫症(GPA、旧名:ウェゲナー肉芽腫症)など、免疫グロブリン投与治療、例えばIVIgが奏功する疾患に関する医療分野において極めて有用である。 The present invention relates to eosinophilic polyangiitis granulosis, which is an infectious disease, an inflammatory disease, idiopathic thrombocytopenic purpura, agammaglobulinemia, acute Kawasaki disease, Guillain-Barre syndrome, and vasculitis Eosinophilic Granulomatosis with Polyangitis: EGPA, formerly known as allergic granulomatous vasculitis, Churg-Strauss syndrome), microscopic polyangiitis (MPA), rapidly progressive glomerulonephritis, diffuse alveolar bleeding due to vasculitis and The present invention is extremely useful in the medical field for immunoglobulin administration therapy, for example, a disease to which IVIg is effective, such as interstitial pneumonia, polyarteritis nodosa, and polyangiitis granulomatosis (GPA, former name: Wegener's granulomatosis).
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